The impact of a pulmonary embolism response team on the efficiency of patient care in the emergency department.

The concept of a pulmonary embolism response team (PERT) is multidisciplinary, with the hope that it may positively impact patient care, hospital efficiency, and outcomes in the treatment of patients with intermediate and high risk pulmonary embolism (PE). Clinical characteristics of a baseline population of patients presenting with submassive and massive PE to URMC between 2014 and 2016 were examined (n = 159). We compared this baseline population before implementation of a PERT to a similar population of patients at 3-month periods, and then as a group at 18 months after PERT implementation (n = 146). Outcomes include management strategies and efficiency of the emergency department (ED) in diagnosing, treating, and dispositioning patients. Before PERT, patients with submassive and massive PE were managed fairly conservatively: heparin alone (85%), or additional advanced therapies (15%). Following PERT, submassive and massive PE were managed as follows: heparin alone (68%), or additional advanced therapies (32%). Efficiency of the ED in managing high risk PE significantly improved after PERT compared with before PERT; where triage to diagnosis time was reduced (384 vs. 212 min, 45% decrease, p = 0.0001), diagnosis to heparin time was reduced (182 vs. 76 min, 58% decrease, p = 0.0001), and the time from triage to disposition was reduced (392 vs. 290 min, 26% decrease, p < 0.0001). Our analysis showed that following PERT implementation, patients with intermediate and high risk acute PE received more aggressive and advanced treatment modalities and received significantly expedited care in the ED.

The impact of a multi-specialty team for high risk pulmonary embolism on resident and fellow education.

The impact of the Pulmonary Embolism Response Team (PERT) model on trainee physician education and autonomy over the management of high risk pulmonary embolism (PE) is unknown. A resident and fellow questionnaire was administered 1 year after PERT implementation. A total of 122 physicians were surveyed, and 73 responded. Even after 12 months of interacting with the PERT consultative service, and having formal instruction in high risk PE management, 51% and 49% of respondents underestimated the true 3-month mortality for sub-massive and massive PE, respectively, and 44% were unaware of a common physical exam finding in patients with PE. Comparing before and after PERT implementation, physicians perceived enhanced confidence in identifying ( p<0.001), and managing ( p=0.003) sub-massive/massive PE, enhanced confidence in treating patients appropriately with systemic thrombolysis ( p=0.04), and increased knowledge of indications for systemic thrombolysis and surgical embolectomy ( p=0.043 and p<0.001, respectively). Respondents self-reported an increased fund of knowledge of high risk PE pathophysiology (77%), and the perception that a multi-disciplinary team improves the care of patients with high risk PE (89%). Seventy-one percent of respondents favored broad implementation of a PERT similar to an acute myocardial infarction team. Overall, trainee physicians at a large institution perceived an enhanced educational experience while managing PE following PERT implementation, believing the team concept is better for patient care.

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts.

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 µg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 µg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 µg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis.

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell-specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation.

Professional, personal and community: 3 domains of physician retention in rural communities.

INTRODUCTION: We sought to explore the professional, personal and community domains of physician retention in 4 rural communities in Alberta and to develop a preliminary framework for physician retention. METHODS: We used a qualitative, collective case study design to study 4 rural communities (cases) in Alberta that retained family physicians for 4 years or longer. Participants included physicians, staff members, spouses and community members. Data collected from interviews, documents and observations were analyzed individually, and similarities and differences across all cases were assessed. RESULTS: A range of factors that could influence physicians' decisions to stay in a particular community were described by participants. Within the professional domain, physician supply, physician dynamics, scope of practice and practice set-up were common across all communities, and innovation, and management and support emerged from some communities. The personal factors, goodness-of-fit, individual choice, and spousal and family support were present in all communities. Four community factors--appreciation, connection, active support, and physical and recreational assets--emerged across all communities, and reciprocity was present in 3 communities. From these data, we developed a preliminary retention framework. CONCLUSION: Physicians, policy-makers and community members are encouraged to consider the 3 retention domains of professional, personal and community.

Phosphorylation of the epidermal growth factor receptor (EGFR) is essential for interleukin-8 release from intestinal epithelial cells in response to challenge with Escherichia coli O157 : H7 flagellin.

Enterohaemorrhagic Escherichia coli O157 : H7 is a major foodborne and environmental pathogen responsible for both sporadic cases and outbreaks of food poisoning, which can lead to serious sequelae, such as haemolytic uraemic syndrome. The structural subunit of E. coli O157 : H7 flagella is flagellin, which is both the antigenic determinant of the H7 serotype, an important factor in colonization, and an immunomodulatory protein that has been determined to be a major pro-inflammatory component through the instigation of host cell signalling pathways. Flagellin has highly conserved N- and C-terminal regions that are recognized by the host cell pattern recognition receptor Toll-like receptor (TLR) 5. Activation of this receptor triggers cell signalling cascades, which are known to activate host cell kinases and transcription factors that respond with the production of inflammatory mediators such as the chemokine interleukin-8 (IL-8), although the exact components of this pathway are not yet fully characterized. We demonstrate that E. coli O157 : H7-derived flagellin induces rapid phosphorylation of the epidermal growth factor receptor (EGFR), as an early event in intestinal epithelial cell signalling, and that this is required for the release of the pro-inflammatory cytokine IL-8.

Physician retention in rural Alberta: key community factors.

OBJECTIVES: As part of a larger case study exploring physician retention factors and strategies employed by rural communities, the objective of this analysis was to explore the community factors that promoted physician retention. METHODS: A qualitative, collective case study design was employed to study four rural communities (cases) in Alberta that retained family physicians for four years or longer. Participants included physicians, staff members, spouses and community members (all were patients from the communities studied). Communities were selected through a retention-specific matrix; each quadrant represented a particular community typology. Case data collected from interviews, documents and observations were analyzed, and similarities and differences among cases were assessed. RESULTS: A range of community factors that could influence physicians' decisions to stay in a particular community were described by participants. Four themes, Appreciation, Connection, Active Support and Physical/Recreational Assets, were positively related to physician retention in the four communities studied. These community factors existed to different degrees but were present in all communities. Reciprocity was a fifth factor that emerged in three of the four communities studied. CONCLUSION: Physicians, policy-makers, community members and health care professionals are encouraged to consider the community domain when planning and implementing strategies to retain rural physicians and other health care professionals. The four communities studied were able to promote retention of their primary care physicians by showing appreciation to them, building connections with them and their families, actively supporting their physicians and local health facilities, maintaining and improving local physical/recreational amenities, and nurturing reciprocal rapport with physicians.

Calnexin phosphorylation: linking cytoplasmic signalling to endoplasmic reticulum lumenal functions.

Calnexin is an abundant integral membrane phosphoprotein of the endoplasmic reticulum (ER) of eukaryotic cells. The role of the luminal domain as an N-glycoprotein specific lectin has been well-established. Cytosolic C-terminal domain phosphorylation of calnexin has recently been elucidated in glycoprotein folding and quality control. Signalling of the presence of unfolded proteins from the lumen of the ER is mediated by the three ER membrane sensor proteins Ire1, ATF6 and PERK. The observation that the C-terminus of calnexin is differentially phosphorylated when glycoproteins are misfolded initiated our search for functional roles of calnexin phosphorylation. Recent studies have defined a role for phosphorylation at a proline-directed kinase site (Ser563) in ER protein quality control, while phosphorylation at a casein kinase 2 site (Ser534, Ser544) may be linked to transport functions. There are also four other abundant integral membrane phosphoproteins in the ER, and these may be components of other signalling pathways that link and coordinate other ER functions with the rest of the cell.

Calnexin phosphorylation attenuates the release of partially misfolded alpha1-antitrypsin to the secretory pathway.

Calnexin is a type I integral membrane phosphoprotein resident of the endoplasmic reticulum. Its intraluminal domain has been deduced to function as a lectin chaperone coordinating the timing of folding of newly synthesized N-linked glycoproteins of the secretory pathway. Its C-terminal cytosolic oriented extension has an ERK1 phosphorylation site at Ser(563) affecting calnexin association with the translocon. Here we find an additional function for calnexin phosphorylation at Ser(563) in endoplasmic reticulum quality control. A low dose of the misfolding agent l-azetidine 2-carboxylic acid slows glycoprotein maturation and diminishes the extent and rate of secretion of newly synthesized secretory alpha1-antitrypsin. Under these conditions the phosphorylation of calnexin is enhanced at Ser(563). Inhibition of this phosphorylation by the MEK1 inhibitor PD98059 enhanced the extent and rate of alpha1-antitrypsin secretion comparable with that achieved by inhibiting alpha-mannosidase activity with kifunensine. This is the first report in which the phosphorylation of calnexin is linked to the efficiency of secretion of a cargo glycoprotein.

Screening methods used to determine the anti-microbial properties of Aloe vera inner gel.

The emergence of antibiotic resistant bacterial strains is a growing problem and is an important concern for patients, physicians, healthcare managers, and policymakers as it results in poorer health and economic outcomes. This has led to an urgent global call for new antimicrobial drugs, particularly from natural resources. We have been studying the antimicrobial properties of the inner leaf gel component of Aloe barbadensis Miller and have used a number of different, simple in vitro assays to establish a scientific basis for the potential use of Aloe vera on a range of clinically relevant bacteria. The bacteria used include Shigella flexneri, Methicillin-Resistant Staphylococcus aureus (MRSA), Enterobacter cloacae and Enterococcus bovis. In this paper, we compare standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI) with a microtitre assay using a metabolic colour indicator Alamar blue. All the techniques described have shown that Aloe vera has an antimicrobial effect, however, the microtitre assay enables high throughput screening, under similar conditions and is less wasteful of plant material.

The inner gel component of Aloe vera suppresses bacterial-induced pro-inflammatory cytokines from human immune cells.

The present study was carried out to examine the anti-inflammatory activity of the inner leaf gel component of Aloe barbadensis Miller. A simple in vitro assay was designed to determine the effect of the inner gel on bacterial-induced pro-inflammatory cytokine production, namely TNF-alpha and IL-1 beta, from peripheral blood leukocytes stimulated with Shigella flexneri or LPS. This report describes the suppression of both cytokines with a freeze-dried inner gel powder and a commercial health drink from the same source. Comparison was made with a human monocytic cell-line (THP-1 cells) and a similar trend in responses was demonstrated.

EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells.

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC.

Conditional expression of MAP kinase phosphatase-2 protects against genotoxic stress-induced apoptosis by binding and selective dephosphorylation of nuclear activated c-jun N-terminal kinase.

MAP Kinase Phosphatase-2 (MKP-2) is a dual specific nuclear phosphatase which is selective for both ERK and JNK, MAP kinases implicated in the regulation of apoptosis in response to genotoxic stress. Here we report the conditional expression of MKP-2 in human embryonic kidney cells 293. We demonstrate that Flag-WT-MKP-2 is able to rescue cells from apoptotic commitment when subjected to UV-C or cisplatin treatment. We establish that upon stimulation all three major MAP kinase families (ERK, JNK and p38 MAP kinases) are activated. However, MKP-2 is surprisingly only able to deactivate JNK in vivo. Furthermore, whilst pre-treatment of cells with either the JNK inhibitor SP600125, or the MEK-1 inhibitor PD98059, also reverses UV-C and cisplatin-induced apoptosis, the anti-apoptotic effect of MKP-2 overexpression is not additive with SP600125 but is with PD098059, suggesting that MKP-2 is involved in specifically terminating JNK activity and not ERK. The inability of MKP-2 to dephosphorylate ERK in vivo is also not due to the inability of Flag-MKP-2 to bind both ERK and JNK; phosphorylated forms of each kinase are co-precipitated with both WT and CI-MKP-2. Immunofluorescence studies however demonstrate that ERK is exclusively cytosolic in origin and not translocated to the nucleus following UV-C and cisplatin treatment whilst JNK is principally nuclear. These studies demonstrate the in vivo specificity of MKP-2 for JNK and not ERK and show that nuclear-targeted JNK is involved in genotoxic stress-induced apoptosis.

The role of intracellular Ca2+ in the regulation of proteinase-activated receptor-2 mediated nuclear factor kappa B signalling in keratinocytes.

1 In this study, we examined the role of Ca2+ in linking proteinase-activated receptor-2 (PAR2) to the nuclear factor kappa B (NFkappaB) pathway in a skin epithelial cell line NCTC2544 stably expressing PAR2 (clone G). 2 In clone G, PAR2-mediated NFkappaB luciferase reporter activity and NFkappaB DNA-binding activity was reduced by preincubation with BAPTA-AM but not BAPTA. Trypsin stimulation of inhibitory kappa B kinases, IKKalpha and IKKbeta, was also inhibited following pretreatment with BAPTA-AM. 3 BAPTA/AM also prevented PAR2-mediated IKKalpha activation in cultured primary human keratinocytes. 4 The effect of BAPTA-AM was also selective for the IKK/NFkappaB signalling axis; PAR2 coupling to ERK, or p38 MAP kinase was unaffected. 5 Pharmacological inhibition of the Ca2+-dependent regulatory protein calcineurin did not inhibit trypsin-stimulated IKK activity or NFkappaB-DNA binding; however, inhibition of Ca2+-dependent protein kinase C isoforms or InsP3 formation using GF109203X or the phospholipase C inhibitor U73122, respectively, reduced both IKK activity and NFkappaB-DNA binding. 6 Mutation of PAR2 within the C-terminal to produce a mutant receptor, which does not couple to Ca2+ signalling, but is able to activate ERK, abrogated NFkappaB-DNA binding and IKK activity stimulated by trypsin. 7 These results suggest a predominant role for the InsP3/Ca2+ axis in the regulation of IKK signalling and NFkappaB transcriptional activation.

Inhibition of lipopolysaccharide-induced macrophage IL-12 production by Leishmania mexicana amastigotes: the role of cysteine peptidases and the NF-kappaB signaling pathway.

Infection with lesion-derived Leishmania mexicana amastigotes inhibited LPS-induced IL-12 production by mouse bone marrow-derived macrophages. This effect was associated with expression of cysteine peptidase B (CPB) because amastigotes of CPB deletion mutants had limited ability to inhibit IL-12 production, whereas preincubation of cells with a CPB inhibitor, cathepsin inhibitor IV, was able to suppress the effect of wild-type amastigotes. Infection with wild-type amastigotes resulted in a time-dependent proteolytic degradation of IkappaBalpha and IkappaBbeta and the related protein NF-kappaB. This effect did not occur with amastigotes of CPB deletion mutants or wild-type promastigotes, which do not express detectable CPB. NF-kappaB DNA binding was also inhibited by amastigote infection, although nuclear translocation of cleaved fragments of p65 NF-kappaB was still observed. Cysteine peptidase inhibitors prevented IkappaBalpha, IkappaBbeta, and NF-kappaB degradation induced by amastigotes, and recombinant CPB2.8, an amastigote-specific isoenzyme of CPB, was shown to degrade GST-IkappaBalpha in vitro. LPS-mediated IkappaBalpha and IkappaBbeta degradation was not affected by these inhibitors, confirming that the site of degradation of IkappaBalpha, IkappaBbeta, and NF-kappaB by the amastigotes was not receptor-driven, proteosomal-mediated cleavage. Infection of bone marrow macrophages with amastigotes resulted in cleavage of JNK and ERK, but not p38 MAPK, whereas preincubation with a cysteine peptidase inhibitor prevented degradation of these proteins, but did not result in enhanced protein kinase activation. Collectively, our results suggest that the amastigote-specific cysteine peptidases of L. mexicana are central to the ability of the parasite to modulate signaling via NF-kappaB and consequently inhibit IL-12 production.

Verotoxin activates mitogen-activated protein kinase in human peripheral blood monocytes: role in apoptosis and proinflammatory cytokine release.

. In this study, we examined the role of mitogen-activated protein (MAP) kinases in the effects of verotoxins (VTs), from Escherichia coli O157:H7, upon both apoptosis and the release of tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulated factor (GM-CSF) from human monocytes. 2. Both VT1 and VT2 stimulated a weak, transient increase in c-Jun-N-terminal kinase (JNK) activity and a strong activation of both p38 mitogen-activated protein kinase (MAP kinase) and extracellular-regulated kinase (ERK) activity in human monocytes, which was sustained in the case of p38 MAP kinase. 3. Stimulation of human monocytes with VT2 (100 ng ml-1) did not result in an increase in apoptosis; however, the toxin stimulated the release of both TNF-alpha and GM-CSF. 4. Pretreatment of human monocytes with the p38 MAP kinase inhibitor SB203580, at concentrations from 100 nM to 10 microM, significantly decreased the VT1- and VT2-induced TNF-alpha and GM-CSF release from monocytes. In contrast, inhibition of MEK1 with PD98059 only significantly decreased GM-CSF release. 5. Pretreatment of monocytes with SP600125 inhibited both GM-CSF and TNF-alpha production; however, significant effects upon p38 MAP kinase and ERK activation were observed. 6. Taken together, these results suggest a role for p38 MAP kinase and ERK in cytokine generation in response to the verotoxins. A role for JNK remains undetermined.

Endosomal proteolysis of glucagon at neutral pH generates the bioactive degradation product miniglucagon-(19-29).

We have investigated the proteolytic mechanisms of glucagon degradation within hepatic endosomes at neutral pH before lumen acidification. Hepatic endosomes incubated at neutral pH rapidly degraded native glucagon into 13 intermediate products, one of which corresponded to the bioactive fragment glucagon-(19-29) (miniglucagon). The serine protease inhibitor phenylmethylsulfonyl fluoride as well as the nonspecific protease inhibitor bacitracin inhibited the endosomal degradation of glucagon at pH 7. In purified endosomal fractions, miniglucagon endopeptidase was undetectable as evaluated by immunoblotting, and immunoprecipitation with antibodies to insulin-degrading enzyme, cathepsins B and D, or furin failed to remove the endosomal neutral glucagonase activity. Incubation of endosomal fractions and [125I]iodoglucagon with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in specific labeling of a 170-kDa polypeptide. The labeling was completely inhibited by unlabeled glucagon (IC50 value, 5 x 10-7 m) and bacitracin (IC50 value, 1 microg/ml), suggesting that it may correspond to a bacitracin-sensitive glucagon-degrading enzyme. Treatment of the 125I-labeled 170-kDa cross-linked polypeptide with N-glycanase demonstrated that the cross-linked complex contained approximately 30 kDa of N-linked oligosaccharides. Specific cross-linking of the 170-kDa polypeptide was also observed using [125I]Tyr12-miniglucagon as the radioligand. Together, these data suggest that the 170-kDa glycoprotein represents a novel glucagon-degrading activity that could mediate glucagon proteolysis within endosomes before the acidification step and generate the bioactive (19-29) miniglucagon peptide.

In vitro susceptibilities of Shigella flexneri and Streptococcus pyogenes to inner gel of Aloe barbadensis Miller.

Aloe barbadensis Miller (or Aloe vera) has widespread use in health products, and despite numerous reports on the whole plant, little work has been performed on the inner gel, which has been used extensively in these products. This report describes the in vitro susceptibilities of two bacteria to this component.

Essential role for verotoxin in sustained stress-activated protein kinase and nuclear factor kappa B signaling, stimulated by Escherichia coli O157:H7 in Vero cells.

The effects of Escherichia coli O157:H7 (strains E30480 and PM601) and the associated verotoxins (VTs), VT1 and VT2, on stress-activated protein kinase and nuclear factor kappa B (NF-kappaB) signaling were investigated with Vero cells, which are extremely sensitive to the cytotoxic effects of E. coli O157:H7 in vitro. Cell-free supernatants prepared from E30480 and PM601 cultures and purified VT1 and VT2 stimulated a strong and prolonged (>4-h) activation of both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase. However, JNK activity stimulated in response to E30480 supernatants was substantially reduced following pretreatment with anti-VT1 and anti-VT2 antibodies, while a VT1 and VT2 gene knockout mutant of PM601 was unable to stimulate JNK activity. E30480 supernatants also caused a sustained activation of NF-kappaB DNA binding, degradation of inhibitory kappa B alpha (IkappaBalpha), and an increase in inhibitory kappa B kinase alpha activity, although PM601 supernatants and VT1 and VT2 had no effect. However, preincubation with VTs prolonged the transient activation of NF-kappaB and IkappaBalpha degradation stimulated by either tumor necrosis factor alpha or interleukin 1beta, while preincubation with anti-VT antibodies prevented the prolonged loss of IkappaBalpha and partially reduced DNA binding in response to E30480 supernatants. These results strongly suggest that in Vero cells, VT plays an essential role in sustained JNK and NF-kappaB signaling in response to E. coli O157:H7 and that this action may underpin their cell-selective cytotoxic effects. These studies also suggest that another component released by strain E30480 contributes to the early activation of JNK and NF-kappaB.